Understanding the normal regulation of the human Gamma globin genes is important for two major reasons: 1) This knowledge will provide new insight into the molecular mechanisms of gene activation and repression during development periods, and 2) knowledge of the normal function of the Gamma genes may allow us to devise new strategies to reactivate these genes in patients with disordersa of the adult Beta globin gene. These studies ae designed to analyze the molecular basis of human Gamma gene activation and repression, by defining the cis and transacting factors that regulate Gamma gene expression. Several laboratories have recently described point mutations approximately 200 nt upstream from the Gamme gene CAP site. These mutations are strongly associated with Hereditary Persistence of Fetal Hemoglobin. One mechanism by which the Gamma globin gene may be activated by these mutations could involve altered binding of regulatory proteins of this region. We have intensively studied the -200 region, and have found that an S1 nuclease hypersensitive site exists here in supercoiled plasmids, and that nuclear extracts from tissue culture cells contain proteins that bind to this region of DNA in vitro. A single nucleotide substitution at position -202 inactivates both protein bidning and the S1 nuclear hypersensitive site. We have also performed a series of experiments designed to determine whether the Gamme gene promoter is under the influene of a tissue-specific enhancer. Initial results indicate that texogenous Gamma gene promoters are enhancer-dependent in a fetal erythroid cell line (K562), implying that the endogenous Gamma gene promoters may also be under the influence of an enhancer. An extensive survey of the Gamma globin gene region for an endogenous enhancer has revealed that a fragment 3' to the human A Gamma globin gene contains an enhancing element. The exact localization of this enhancing element, and its possible function in vivo, are currently under active investigation.